Biochemical identification of bacteria

Various microorganisms show great differences in the types of metabolism. This difference is more pronounced due to the unique single-cell prokaryotic properties of bacteria. Different bacteria have different enzyme systems, so different bacteria have different abilities to decompose and utilize carbohydrates, fats and protein substances. They have different metabolic profiles and decomposition products, and use this feature to establish bacterial biochemical reactions. The classification and identification of strains have major significance.

【Purpose】

1. Understand the physiological and biochemical test reaction principles commonly used in bacterial identification.

2. Master the techniques and methods for determining the physiological and biochemical reactions of bacteria.

[Experimental principle]

Various bacteria have different enzyme systems, so that they can utilize different substrates, or although different substrates can be utilized to produce different metabolites, various physiological and biochemical reactions can be utilized to identify bacteria.

[Reagents and equipment]

1. Strain of Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, Bacillus subtilis

2. Medium glucose protein hydrophobic medium, peptone water medium, sugar fermentation medium (glucose, lactose or sucrose)

3. Reagent 40% NaOH solution, creatine, methyl red reagent, guanidine reagent, 8, 1.6% bromomethylphenol purple indicator.

4. Ultra-clean instrument table, constant temperature incubator, autoclave, test tube, pipette, Du's small casing.

5. Process sugar fermentation test → VP test → methyl red test → 吲哚 test.

[Experimental methods and steps]

Sugar fermentation experiment

1 Test tube labeled with different fermentation broth (such as glucose, sucrose and lactose) fermentation tubes, according to the bacteria needed to be labeled (such as Escherichia coli, Enterobacter aerogenes, common proteobacteria, etc. Blank control).

2 Inoculation culture A small amount of lawn moss was inoculated into the corresponding test tubes by aseptic operation. The blank control was not inoculated, and cultured in a 37 ° C incubator, and the results were observed at 24 h, 48 h and 72 h, respectively.

3 Observation and record After fermentation, compared with the blank control, if the inoculum culture medium maintains the original color, the reaction result is negative, indicating that the bacteria can not use the sugar, the record is indicated by "-"; if the culture medium is yellow The reaction result was positive, indicating that the bacteria can decompose the sugar to produce acid, and the record is represented by "+". The bubble in the Du's tube in the culture solution is positive, indicating that the bacteria can produce acid and produce gas, and the record is represented by "+"; if there is no bubble in the Du's tube, the record is represented by "-".

2. VP test

1 Label the tube and take the tube containing the glucose peptone broth, and mark the cultured bacteria as needed.

2 Inoculation culture Inoculate a small amount of lawn moss into the above corresponding tubes in aseptic operation. The blank control tube is not inoculated, and placed in a 37 ° C incubator for 24 to 48 hours.

3 Observation Record The above tube was taken out and shaken for 2 min. In addition, take 5 empty test tubes corresponding to the bacterial name, add 3 ~ 5ml of the corresponding medium in the corresponding tube, then add 10 ~ 20 drops of 40% NaOH solution, and add about 0.5 ~ 1mg of micro creatine, shake the test tube, so that The oxygen in the air is dissolved and placed in a 37 ° C incubator for 15 to 30 minutes. If the culture solution is red, it is recorded as a positive reaction of VP test (indicated by "+"); if it is not red, it is recorded as a negative reaction of VP test. (indicated by "-").

Note: The culture solution left in the original tube is used as a methyl red test.

3. Methyl red test (MR test)

Take the test tube containing the glucosinolate water culture solution, inoculate the bacteria to be cultured and identified. After night culture, add 2 to 3 drops of methyl red indicator. Pay attention to the addition along the tube wall, carefully observe the upper layer of the culture solution, if the upper layer of the culture solution It turns red, which is a positive reaction; if it is still yellow, it is a negative reaction, which is represented by "+" or "-" respectively.

4. 吲哚 test

1 Test tubes are labeled with a test tube containing peptone water culture solution, and the bacteria to be cultured are labeled.

2 Inoculation culture Inoculate a small amount of lawn moss into the corresponding test tube by aseptic operation, and the fifth tube is not inoculated as a blank control, and cultured in a 37 ° C incubator for 24 to 48 hours.

3 Observation Record Add 5 to 10 drops of sputum reagent to the culture solution, and float the reagent on the surface of the culture to form two layers. Observe the result: if there is sputum, the layer will show rose red, which is a positive test of sputum test. Otherwise, it is a negative reaction, positive for "+" and negative for "-".

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